Due to broad application of probiotics, research efforts are diluted, thus contributing to rejection of health claims. This study aimed to systematically prioritize the unmet needs and research opportunities in infants, children, adults and elderly for probiotics according to their relative importance from a key-opinion-leader (KOL) perspective. In addition, it reveals potential probiotic product characteristics that need improvement from both a patient or consumer and a KOL perspective. Moreover, KOL involvement in the probiotic research & development (R&D) process was assessed. Data was collected by conducting semi-structured interviews and subsequent online questionnaires. The three clinical indications requiring the highest research attention for infants were (infectious) diarrhoea, antibiotic-associated diarrhoea (AAD) and necrotizing enterocolitis (NEC); for children, obesity, AAD and diarrhoea and for adults, AAD, irritable bowel syndrome (IBS) and Alzheimer's disease. From both a KOL as well as a patient or consumer perspective, clinical evidence needs to be improved for probiotic products. Although the majority of the KOLs consult for and perform research with probiotic companies, they are (surprisingly) not able to sufficiently influence industry research agendas. This study incorporates the insights of KOLs and provides direction in the R&D of probiotics to fulfil unmet medical needs in patients.